Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes

He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.     

Molecular characterisation of the pericentric inversion that distinguishes human chromosome 5 from the homologous chimpanzee chromosome

Human and chimpanzee karyotypes differ by virtue of nine pericentric inversions that serve to distinguish human chromosomes 1, 4, 5, 9, 12, 15, 16, 17, and 18 from their chimpanzee orthologues. In this study, we have analysed the breakpoints of the pericentric inversion characteristic of chimpanzee chromosome 4, the homologue of human chromosome 5. Breakpoint-spanning BAC clones were identified from both the human and chimpanzee genomes by fluorescence in situ hybridisation, and the precise locations of the breakpoints were determined by sequence comparisons. In stark contrast to some other characterised evolutionary rearrangements in primates, this chimpanzee-specific inversion appears not to have been mediated by either gross segmental duplications or low-copy repeats, although micro-duplications were found adjacent to the breakpoints. However, alternating purine–pyrimidine (RY) tracts were detected at the breakpoints, and such sequences are known to adopt non-B DNA conformations that are capable of triggering DNA breakage and genomic rearrangements. Comparison of the breakpoint region of human chromosome 5q15 with the orthologous regions of the chicken, mouse, and rat genomes, revealed similar but non-identical syntenic disruptions in all three species. The clustering of evolutionary breakpoints within this chromosomal region, together with the presence of multiple pathological breakpoints in the vicinity of both 5p15 and 5q15, is consistent with the non-random model of chromosomal evolution and suggests that these regions may well possess intrinsic features that have served to mediate a variety of genomic rearrangements, including the pericentric inversion in chimpanzee chromosome 4.     

Molecular Definition of Pericentric Inversion Breakpoints Occurring during the Evolution of Humans and Chimpanzees

High-resolution G-banding analysis has demonstrated remarkable morphological conservation of the chromosomes of the Hominidae family members (humans, chimpanzees, gorillas, and orangutans), with the most notable differences between the genomes appearing as changes in heterochromatin distribution and pericentric inversions. Pericentric inversions may have been important for the establishment of reproductive isolation and speciation of the hominoids as they diverged from a common ancestor. Here the previously published primate karyotype comparisons, coupled with the resources of the Human Genome Project, have been used to identify pericentric inversion breakpoints seen when comparing the human karyotype to that of chimpanzee. Yeast artificial chromosome (YAC) clones were used to detect, by fluorescencein situhybridization, five evolutionary pericentric inversion breakpoints present on the chimpanzee chromosome equivalents of human chromosomes 4, 9, and 12. In addition, two YACs from human 12p that detect a breakpoint in chimpanzee detect a similar rearrangement in gorilla.     

Alpha and beta heterochromatin in polytene chromosome 2 ofDrosophila melanogaster

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization with Rsp sequences (H _o clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed. Received: 17 November 1995; in revised form: 10 April 1996 / Accepted: 18 September 1996     

Structural variability of human chromosome 9 in relation to its evolution

Summary Human chromosome 9 shows a high susceptibility for structural rearrangements, particularly pericentric inversions, which often are transmitted. Three types of pericentric inversions can be observed on No. 9: 1) Type I, showing the total constitutive heterochromatin in the short arm. 2) Type II with part of the C heterochromatin on the short arm, the rest located on the long arm proximal to the centromere. 3) Type III: a subtelocentric chromosome with part of the C heterochromatin in the very short arm and the rest located interstitially on the long arm. With these inversions as well as with other structural rearrangements, e.g. translocations, the break-points are located preferentially within the C heterochromatin or close to the heterochromatic-euchromatic junctions. These findings are in contrast to the findings in lymphocytes from 5 patients with Fanconi's anemia and after irradiation in vitro, reported in the literature. In lymphocytes break-points seem to be distributed more or less by chance. These observations together led us to speculate that human chromosome 9 primarily was an acrocentric chromosome; in morphology and at least in some functions similar to D-and G-group chromosomes. During evolution this acrocentric chromosome changed to a submetacentric one due to a pericentric inversion.The author is sponsored by the Deutsche Forschungsgemeinschaft.     

Origin and Differentiation of a Sex Chromosome System in Parodon hilarii (Pisces, Parodontidae). Satellite DNA, G- and C-banding

A satellite DNA sequence of Parodon hilarii (named pPh2004) was isolated, cloned and sequenced. This satellite DNA is composed of 200 bp, 60% AT rich. In situ hybridization (FISH) results revealed that the satellite DNA pPh2004 is located in the terminal regions of several chromosomes, forming highly evident blocks in some and punctual marks in others. The comparison between the FISH and C-banding results showed that the location of this satellite DNA coincides with that of most terminal heterochromatins. However, some regions are only marked by FISH whereas other regions are only marked by C-banding. The possible existence of more than one satellite DNA family could explain these partial differences. The in situ hybridization with the satellite DNA and the G- and C-bandings confirmed the presence of a sex chromosome system of the ZZ/ZW type in P. hilarii, as well as the correct identification of the Z chromosome in the karyotype. This chromosome displays a segment of terminal heterochromatin in the long arm, similar to the segment observed in the short arm of the W chromosome, also showing a G-banding pattern similar to that of the short arm and part of the long arm of the W chromosome. A hypothesis on the origin of the W chromosome from an ancestral chromosome similar to the Z chromosome is presented.     

Mammalian ChlR1 has a role in heterochromatin organization

The ChlR1 DNA helicase, encoded by DDX11 gene, which is responsible for Warsaw breakage syndrome (WABS), has a role in sister-chromatid cohesion. In this study, we show that human ChlR1 deficient cells exhibit abnormal heterochromatin organization. While constitutive heterochromatin is discretely localized at perinuclear and perinucleolar regions in control HeLa cells, ChlR1-depleted cells showed dispersed localization of constitutive heterochromatin accompanied by disrupted centromere clustering. Cells isolated from Ddx11^−/− embryos also exhibited diffuse localization of centromeres and heterochromatin foci. Similar abnormalities were found in HeLa cells depleted of combinations of HP1α and HP1β. Immunofluorescence and chromatin immunoprecipitation showed a decreased level of HP1α at pericentric regions in ChlR1-depleted cells. Trimethyl-histone H3 at lysine 9 (H3K9-me3) was also modestly decreased at pericentric sequences. The abnormality in pericentric heterochromatin was further supported by decreased DNA methylation within major satellite repeats of Ddx11^−/− embryos. Furthermore, micrococcal nuclease (MNase) assay revealed a decreased chromatin density at the telomeres. These data suggest that in addition to a role in sister-chromatid cohesion, ChlR1 is also involved in the proper formation of heterochromatin, which in turn contributes to global nuclear organization and pleiotropic effects.     

Direct evidence for suppression of recombination within two pericentric inversions in humans: a new sperm-FISH technique.

Crossover within a pericentric inversion produces reciprocal recombinant chromosomes that are duplicated/deficient for all chromatin distal to the breakpoints. In view of this fact, a new technique is presented for estimating the frequency of recombination within pericentric inversions. YAC probes were selected from within the q- and p-arm flanking regions of two human inversions, and two-color FISH analysis was performed on sperm from heterozygous inversion carriers. A total of 6,006 sperm were analyzed for chromosome 1 inversion (p31q12), and 3,168 were analyzed for chromosome 8 inversion (p23q22). Both inversions displayed suppression of crossing-over, although the amount of suppression differed between the two inversions. The recombination frequency of 13.1% recorded for chromosome 8 inversion was similar to the frequency of 11.4% previously estimated by the human/hamster-fusion method. For chromosome 1 inversion, the recombination frequency of 0. 4% reported here was below the limits of detection of the fusion technique. The simplicity of the FISH technique and the ease of scoring facilitate analysis of a sample-population size much larger than previously had been possible.     

Characterization of the human lineage-specific pericentric inversion that distinguishes human chromosome 1 from the homologous chromosomes of the great apes

The human and chimpanzee genomes are distinguishable in terms of ten gross karyotypic differences including nine pericentric inversions and a chromosomal fusion. Seven of these large pericentric inversions are chimpanzee-specific whereas two of them, involving human chromosomes 1 and 18, were fixed in the human lineage after the divergence of humans and chimpanzees. We have performed detailed molecular and computational characterization of the breakpoint regions of the human-specific inversion of chromosome 1. FISH analysis and sequence comparisons together revealed that the pericentromeric region of HSA 1 contains numerous segmental duplications that display a high degree of sequence similarity between both chromosomal arms. Detailed analysis of these regions has allowed us to refine the p-arm breakpoint region to a 154.2 kb interval at 1p11.2 and the q-arm breakpoint region to a 562.6 kb interval at 1q21.1. Both breakpoint regions contain human-specific segmental duplications arranged in inverted orientation. We therefore propose that the pericentric inversion of HSA 1 was mediated by intra-chromosomal non-homologous recombination between these highly homologous segmental duplications that had themselves arisen only recently in the human lineage by duplicative transposition.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .Justyna M. Szamalek and Violaine Goidts are contributed equally to the paper.     

Characterization and Comparative Analysis of DNA from the Pericentric Heterochromatin of Chromosome 2 of Anopheles atroparvus V. Tiel (Culicidae, Diptera)

The library containing DNA sequences from the diffuse pericentric heterochromatin from the right arm ofAnopheles atroparvus V. Tiel (Culicidae, Diptera) chromosome 2 (2R) was generated by use of chromosome microdissection technique. Southern-blot hybridization of the library fragments with the labeled genomic DNA of A. atroparvus and analysis of their primary structure showed that this heterochromatin region contained repeated DNA sequences differed by their primary structure and the number of copies. These were mostly AT-rich sequences harboring the features characteristic of the S/MAR regions. Based on the clones homology to the sequences from the A. gambiae and Drosophila melanogaster genomes, it was demonstrated that the pericentric heterochromatin from the right arm of A. atroparvus chromosome 2 contained gypsy-like transposable elements, as well as the sequences homologous to the structural genes. In situ hybridization with the chromosomes of A. atroparvus and of the two representatives of the Anopheles maculipennis species complex, A. messeae and A. beklemishevi, showed that pericentric regions of all these chromosomes contained DNA sequences homologous to the sequences from the region-specific library. Cloned fragments of conserved repetitive DNA revealed upon interspecific Southern-blot hybridization of the clones with the labeled genomic DNA of A. messeae can be utilized in further investigations of evolutionary rearrangements of the pericentric heterochromatin within the Anopheles maculipennis species complex.     

Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH

Human blood leukocytes within an agarose matrix were deproteinized and exposed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile sites. Since the amount of ssDNA produced within a specific sequence area may be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FISH). The DBD-FISH signal, corrected for the respective FISH signals in metaphase, was remarkably strong in the 5bp classical satellite DNA domains analyzed (D1Z1, D9Z3, DYZ1), intermediate in the classical satellite 1 DNA sequences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromeres). This result is evidence of a high density of constitutive alkali-labile sites, probably abasic sites, within the 5bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labile sites could explain the high frequency of spontaneous breakage and rearrangements in pericentromeric heterochromatin of chromosomes 1, 9, and 16, but not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment.     

Pericentric inversions in man: personal experience and review of the literature

Summary The Leuven cytogenetic centre experience on pericentric inversion in man is discussed with exclusion of the pericentric inversions of the heterochromatic blocks of chromosomes 1 and 9. In a total of 51,500 patients, referred for constitutional chromosome analysis during the period 1970–1985, pericentric inversions were found in 24 index patients. The breakpoints detected in these different pericentric inversions are summarized and compared to those found in previous reports. Bands 2p13, 2q21, 5q31, 6c21, 10q22, and 12q13 were shown to be repeatedly involved in the different studies and, furthermore, breakpoints at bands 2q11, 5p13, 5p15, 5q13, 7q11, 11q25, and 14p11 were present in this study as well as in our previous review on reciprocal autosomal translocations. In 13 familial pericentric inversions, even after exclusion of all inversion carrier probands, a 1.6:1 excess of pericentric inversion carriers versus karyotypically normal progeny was observed. While chromosomally unbalanced offspring represent 3.5% of all chromosomally investigated liveborns of the present study, 7.1% of all liveborn inversion carrier offspring presented with a mental retardation and/or multiple congenital anomalies (MR/MCA) problem. Additional chromosomal abnormalities, i.e. a 21 trisomy and an accessory small ring chromosome were observed in two pericentric inversion carriers. These data and results are discussed and compared to the data available in the literature.     

Nonrandom chromosomal distribution of spontaneous breakpoints and satellite DNA clusters in two geographically distant populations of Chironomus riparius (Diptera: Chironomidae)

Two geographically distant populations of Chironomus riparius (syn. C. thummi) from two environmentally polluted sites (Santena, Italy and Varna, Bulgaria) show numerous somatic and inherited chromosomal aberrations (inversions, deletions and deficiencies). Fifty-five percent of the observed breakpoints occurred in at least two larvae from both populations. Breakpoints occurring twice or more were considered as common structural chromosomal breakpoints. We tested whether such common breakpoints in larvae of the two polluted populations had a random chromosomal distribution or occurred preferentially in specific heterochromatic regions. Distribution of common breakpoints was not random, and proximal regions of first and third chromosome had significantly more common breakpoints than distal ones. By FISH we identified and mapped 56 chromosomal sections containing clusters of two tandem-repetitive satellite DNA families called Hinf and Alu elements. Like the common breakpoints, these repetitive DNA clusters appeared to be significantly more abundant in regions of constitutive heterochromatin such as the pericentromeric regions, while in distal sections of chromosomal arms they were rare or absent. Twenty-four out of 45 common breakpoints (i.e., 53.3%) occurred in cytogenetic sections where Alu and Hinf satellite DNA probes hybridized. The frequency of co-localization between common breakpoints and repetitive DNA hybridization signals was significantly higher than expected by chance. We hypothesize that spontaneous or induced breaks occur more frequently in sections containing blocks of repetitive DNA.     

Evidence for two successive pericentric inversions in sex lampbrush chromosomes of Rana rugosa (Anura: Ranidae)

The objective of this study was to clarify the course of inversions by which a ZW sex chromosome dimorphism has become established in Rana rugosa. Fortunately, R. rugosa preserves three different forms of sex chromosomes in the several isolated populations. In both males and females, the homomorphic sex chromosomes from Hiroshima were closely similar to Z, while those from Isehara were slightly different from the Z. Females from Hirosaki demonstrated heteromorphic sex chromosomes. In this study, the configuration and pairing behavior of sex lampbrush chromosomes were examined in the female offspring produced from a cross between a female from Hiroshima and a male from Isehara, as well as the female offspring of a female from Hirosaki and the male from Isehara. For the sex lampbrush chromosomes from Hiroshima and Isehara, chiasmata were exclusively formed between the distal regions of the long arms of one sex chromosome and the terminal regions of the short arms of the other. As a result, landmarks arranged in reverse order were observed in the achiasmatic regions of these chromosomes. For the sex lampbrush chromosomes from Isehara and Hirosaki, on the other hand, chiasma formation was mainly confined to the lower half of the chromosomes corresponding to the long arms, and the landmarks in the achiasmatic regions of these chromosomes were disposed in the opposite direction to each other. These results seem to indicate that in the primitive sex chromosomes of the Hiroshima type two pericentric inversions occurred, leading to the differentiation of the W chromosomes. This is the first report to substantiate the process of sex chromosome differentiation experimentally. Received: 10 November 1996; in revised form: 22 April 1997 / Accepted: 24 April 1997     

Radiation-induced DNA breaks in different human satellite DNA sequence areas,analyzed by DNA breakage detection-fluorescence in situ hybridization

Human blood leukocytes were exposed to X rays to analyze the initial level of DNA breakage induced within different satellite DNA sequence areas and telomeres, using the DNA breakage detection-FISH procedure. The satellite DNA families analyzed comprised alphoid sequences, satellite 1, and 5-bp classical satellite DNA sequences from chromosome 1 (D1Z1 locus), from chromosome 9 (D9Z3 locus), and from the Y chromosome (DYZ1 locus). Since the control hybridization signal was quite different in each of the DNA targets, the relative increase in whole fluorescence intensity with respect to unirradiated controls was the parameter used for comparison. Irradiation of nucleoids obtained after protein removal demonstrated that the alkaline unwinding solution generates around half the amount of signal when breaks are present in the 5-bp classical DNA satellites as when the same numbers of breaks are present the genome overall, whereas the signal is slightly stronger when the breaks are within the alphoids or satellite 1 sequences. After correction for differences in sensitivity to the alkaline unwinding-renaturation, DNA housed in chromatin corresponding to 5-bp classical satellites proved to be more sensitive to breakage than the overall genome, whereas DNA in the chromatin corresponding to alphoids or satellite 1 showed a sensitivity similar to that of the whole genome. The minimum detectable dose was 0.1 Gy for the whole genome, 0.2 Gy for alphoids and satellite 1, and 0.4 Gy for the 5-bp classical satellites. Telomeric DNA sequences appeared to be maximally labeled in unirradiated cells. Thus telomeric ends behave like DNA breaks, constituting a source of background in alkaline unwinding assays.     

STRUCTURE AND POPULATION GENETICS OF THE BREAKPOINTS OF A POLYMORPHIC INVERSION IN DROSOPHILA SUBOBSCURA

Drosophila subobscura is a paleartic species of the obscura group with a rich chromosomal polymorphism. To further our understanding on the origin of inversions and on how they regain variation, we have identified and sequenced the two breakpoints of a polymorphic inversion of D. subobscura—inversion 3 of the O chromosome—in a population sample. The breakpoints could be identified as two rather short fragments (～300 bp and 60 bp long) with no similarity to any known transposable element family or repetitive sequence. The presence of the ～300‐bp fragment at the two breakpoints of inverted chromosomes implies its duplication, an indication of the inversion origin via staggered double‐strand breaks. Present results and previous findings support that the mode of origin of inversions is neither related to the inversion age nor species‐group specific. The breakpoint regions do not consistently exhibit the lower level of variation within and stronger genetic differentiation between arrangements than more internal regions that would be expected, even in moderately small inversions, if gene conversion were greatly restricted at inversion breakpoints. Comparison of the proximal breakpoint region in species of the obscura group shows that this breakpoint lies in a small high‐turnover fragment within a long collinear region (～300 kb).     

Evolution versus constitution: differences in chromosomal inversion

We compared the chromosomal breakpoints of evolutionary conserved and constitutional inversions. Multicolor banding and human-specific bacterial artificial chromosomes were applied to map the breakpoints of constitutional pericentric inversions on human chromosomes 2 and 9. For the first time, we present a high-resolution analysis of the breakpoint regions, which are characterized by gene destitution, co-localization with fragile sites, multitude repeats as well as pseudogenes and, remarkably, a large sequence homology to the opposite breakpoint. In contrast, evolutionary inversion breakpoints lack such extensive cross-hybridizing regions and are often associated with fragile sites of the genome and low-copy repeats. These molecular characteristics gave evidence for different types of inversion formation and indicate that evolutionary inversions cannot originate from constitutional inversions like those of chromosomes 2 and 9. Finally, the constitutional inversion breakpoints were investigated on three different great ape species and on four test persons each bearing the same cytogenetically determined inversion on chromosomes 2 and 9, respectively. Our data indicate the existence of different molecular breakpoints for the two variant chromosomes.     

Production and identification of new structural chromosome mutations in barley (Hordeum vulgare L.)

A total of 52 reciprocal translocations and 9 pericentric inversions were induced and identified in both standard and cytologically marked barley karyotypes using gamma-rays as the clastogenic agent. An analysis based upon Giemsa N-banding patterns and arm length measurements of the reconstructed chromosomes enabled a rather precise cytological localization of intra- and interchange breakpoints. This analysis was significantly facilitated and improved, especially for the identification of pericentric inversions, when the reconstructed karyotype T-1586 was used as starting material. The majority, if not all, of the aberration breakpoints proved to be localized in interband regions or in medial and terminal parts of the chromosomes, i.e., in regions which are deficient in constitutive heterochromatin. A great number of the structural mutations produced in this study contain specific cytological markers covering nearly all of the chromosomes of barley karyotype. This material might be of considerable interest in solving various problems of barley cytogenetics and chromosome engineering and especially in constructing a physical map of barley genome.